J. Plant Physiol. 160.

1065 1071 (2003)

Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp

Influence of temperature on biomass, iron metabolism and some related

bioindicators in tomato and watermelon plants
Rosa M. Rivero, Esteban Snchez, Juan M. Ruiz, Luis Romero*
Department of Plant Biology, Faculty of Sciences, University of Granada, 18071-Granada, Spain
Received August 8, 2002 Accepted December 5, 2002

Summary
Tomato, Lycopersicon esculentum L. cv. RX-335, and watermelon plants, Citrullus lanatus [Thom.]
Mansf. cv. F-90 were grown under controlled conditions at three different temperatures (10, 25 and
35 C) for 30 days. The aim of the experiment was to analyse the effect of the different temperatures
on Fe uptake and distribution, as well as the behaviour of the main bioindicators of this element.
Thus, we analysed the total and free Fe concentrations and H2O2 concentrations, as well as enzymatic activities of Fe-chelate reductase (FeCH-R), aconitase (Aco), guaiacol peroxidase (GPX), catalase (CAT), and Fe-superoxide dismutase (FeSOD), and the dry weight of the plants. The effect
caused by each temperature varied according to the species of plant. Our results indicate that heat
stress appears in tomato plants when grown at 35 C (above the optimal temperature for growth),
while in watermelon plants, which need more heat than do tomatoes, cold stress appears at 10 C
(below the optimal temperature for growth). Despite these differences between the two species, the
results under conditions of thermal stress were the same: 1) decreased shoot weight, 2) reduced Fe
uptake, 3) depressed activities of FeCH-R, Aco, GPX, CAT and 4) boosted SOD activity. In short, our
results appear to indicate that, whether heat in tomato plants or cold stress in watermelon plants, Fe
uptake was diminished, as were the enzymatic activities related to the levels of this micronutrient in
the plant. The high FeSOD activity in these plants could be explained by a defensive response to
heat or cold stress.
Key words: Bioindicators temperature Fe-metabolism tomato watermelon
Abbreviations: Aco = aconitase. CAT = catalase. EDTA = ethylenediaminetetraacetic acid.
FeCH-R = Fe(III)-chelate reductase. FeSOD = ferro-superoxide dismutase. GPX = guaiacol peroxidase. H2O2 = hydrogen peroxide. NBT = nitro blue tetrazolium. PVPP = polyvinylpolypyrrolidone

Introduction
During the normal processes of growth and development,
plants are subjected to different types of stress, such as
* E-mail corresponding author: lromero@ugr.es

drought, heat, ultraviolet light, air pollution and pathogen

attack (McKersie and Leshem 1994, Paliyath and Fletcher
1995, Paliyath et al. 1997). In many plants, physiological and
biochemical alterations accur after exposure to temperatures
higher or lower than for optimal growth (Grace et al. 1998).
The results of these disturbances, which are reflected in most
0176-1617/03/160/09-1065 $ 15.00/0

1066

Rosa M. Rivero et al.

metabolic processes (Anderson et al. 1994, Prasad et al.

1994 a, b), may be a reduced growth capacity of crops and
therefore lower commercial yield (Wang 1982).
Plants have an ideal temperature range for optimum
absorption of mineral nutrients (Hamlin et al. 1999), which is
an important aspect of plant growth and development (Briat
and Lobraux 1997). Iron is an essential cation because of its
physico-chemical properties: coordinated at metalloprotein
active sites, it participates in most of basic redox reactions
required in both the production and consumption of oxygen.
Iron also is involved in many vital enzymatic reactions
required for nitrogen fixation, DNA synthesis (ribonucleotide
reductase) and hormone synthesis (lipoxygenase and ACC
oxidase) (Briat and Lobraux 1997). It had been suggested
that the temperature affected Fe absorption. Hamlin et al.
(1999) found that pansy shoot contained more Fe at 22 that
at 12 C. These results are similar to those reported in tomato,
which contained increasing amounts of foliar Fe as temperature increased (12 to 24 C, Gosselin and Trudel 1983). On
the other hand, FeCH-R activity is partially inhibited at extreme temperatures, causing less reduction of Fe3 + to Fe2 +
and diminishing its plant availability (Hamlin et al. 1999).
Because little information is available on heat and cold
stress in relation to the nutritional status of Fe and its principal
bioindicators, this experiment was designed to evaluate the
effect of temperature in tomato and watermelon plants on biomass, Fe uptake and some bioindicators.

Material and Methods

Plant material and culture conditions
Tomato (Lycopersicon esculentum L. cv. RX-335) and watermelon
(Citrullus lanatus [Thomb.] Mansf. cv. Dulce maravilla) plants were
germinated and grown for 30 days in growth chambers at optimal
growth temperatures for each species: 22 26 C for the tomato (Veschambre and Zuang 1979) and 33 37 C for the watermelon (Wolf
1982, Maroto 1995). Thereafter, 12 plants per species were transferred
to each of three cultivation chambers set at 10 C (day/night), 25 C
(day/night) and 35 C (day/night). Each temperature experiment was
conducted for 30-days. The growth chambers in all cases were maintained at a relative humidity of 60 80 % and 16 h of photoperiod at a
PPFD of 350 mol m 2 s 1 (measured at the top of the plants with a 190
SB quantum sensor, LI-COR Inc., Lincoln, EN, USA).
During all experiments the plants were grown in individual pots
(25 cm upper diameter, 17 cm lower diameter, 25 cm in height) filled
with vermiculite and received a nutrient solution of: 2 mmol/L KNO3,
4 mmol/L (NO3)2Ca, 1.5 mmol/L NaH2PO4, 2 mmol/L CaCl2, 3 mmol/L
SO4K2, 1.25 mmol/L MgSO4, 50 mol/L Fe-EDTA, 2 mol/L MnSO4,
1 mol/L ZnSO4, 0.25 mol/L CuSO4, 0.05 mol/L (NH4)6Mo7O24 and
25 mol/L H3BO3 (van Zinderen 1986). The nutrient solution (pH 6 to
6.1) was renewed every 3 days.
Plants were sampled on day 60 after sowing, all sampled leaves
being at the mature state. The material was rinsed three times in deionised H2O after disinfecting with 1 % non-ionic detergent (Wolf 1982)
and then blotted on filter paper. A subsample of roots and leaves were

used fresh for the analysis of FeCH-R, GPX, CAT, Aco and FeSOD,
performing triplicate assays for each extraction. A subsample of roots
and leaves of the plants were dried in a forced-air oven at 70 C for
24 h and utilised for quantification of dry weight of plants.

Plant analysis
Extraction and assay of FeCH-R
The extraction and assay methods for fresh roots of tomato and watermelon plants used were those proposed by Brggemann and Moog
(1989). Plasma membrane vesicles were prepared from the 10,000
30,000 g pellet using an aqueous two-phase system. The reaction
was started by the addition of reaction mixture (50 L Tris-MES
20 mmol/L pH 7.0, 50 L MgCl2 5 mmol/L, 50 L Triton X-100 0.02 % (v/
v), 50 L Fe3 + 500 mol/L, 50 L BPDS 500 mol/L, 50 L NADH
500 mol/L) of 50 L of the membrane-containing solution and proceeded in the dark for 30 min. Controls (without membranes) and
blanks (without Fe3 + -EDTA or NADH) were treated the same way. After
30 min, the concentration of the Fe2 + (BPDS)3 complex was measured
photometrically at 535 nm. Triplicate assays were performed for each
extract.

Extractions and assays of GPX and CAT

Extraction and assay of GPX activity were carried out as described
Kalir et al. (1984) and Ruiz et al. (1998) by the oxidation of guaiacol in
the presence of H2O2 (extinction coefficient, 26.6 mmol/L 1 cm 1) at
470 nm. Extraction and assay of CAT activity were determined as
described Badiani et al. (1990) by following the consumption of H2O2
(extinction coefficient, 39.4 mmol/L 1 cm 1) at 240 nm for three minutes.

Extraction and assay of Aco

Extraction and assay of Aco activity were carried out as described by
De Bellis et al. (1993) by following the formation of cis-aconitic acid at
240 nm for three minutes.

Extraction and assay of FeSOD

Extraction and assay of FeSOD activity were carried out as described
Beyer and Fridocvitch (1987) with some modifications (Yu et al. 1998)
following the inhibition of photochemical reduction of nitro blue tetrazolium (NBT). The reaction mixtures were illuminated for 15 min with a
light intensity of 380 mol m 2 s 1. Identical reaction mixtures that were
not illuminated were used to correct for background absorbance. One
unit of FeSOD activity was defined as the amount of enzyme required
to cause 50 % inhibition of the reduction of NBT as monitored at
560 nm. To determine whether the reaction was enzymatic, the sample
extract was boiled and assayed. Protein was estimated by the method
of Bradford (1976).

Estimation of total and free Fe

For the determination of total Fe, dry root and leaf weight (0.1 g) were
submitted to a sulphuric-acid digestion in presence of H2O2 (Wolf

Effect of temperature on Fe metabolism

1982), and diluted with double distilled water. Total Fe was analysed
by atomic-absorption spectrophotometry (Hocking and Pate 1977).
The free Fe was analysed from an extraction of 0.2 g of dried and
ground root and leaf material in 10 mL of 1 N HCl (Hocking and Pate
1977).

Estimation of H2O2 concentration

The methods used for extraction and quantification of total H2O2 were
those of MacNevin and Uron (1953) and Brennan and Frenkel (1977).
Hydroperoxides form a specific complex with titanium (Ti + 4), which
can be measured by colorimetry at 415 nm. The concentration of peroxide in the extracts was determined by comparing the absorbance
against a standard curve representing a titanium-H2O2 complex from
0.1 to 1mmol/L. The hydroperoxides represent the total peroxides.

Statistical analysis
Standard analysis of variance was used to assess the significance of
treatment. Results shown are mean values SE. A correlation analysis
was also conducted to determine the relations between the different
variables. Levels of significance are represented by at * P < 0.05, ** at
P < 0.01, *** at P < 0.001 and ns: not significant by ANOVA at P = 0.05.
Only differences between the two temperatures were tested and separated ANOVAs were conducted for each variable. Finally, t-test and
Bonferroni correction were made for each variable.

Results and Discussion

Iron metabolism in tomato plants
Tomato plant requires optimal temperatures of 22 26 C for
growth and development (Brinen 1979, Maroto 1995). Under
the experimental conditions, the highest values for shoot dry
weight were registered at 25 C (optimal temperature) and the
lowest at 35 C (Fig. 1, P < 0.001), representing a 52 % reduction. Similar results were found by Hood and Mills (1994) in

1067

the cultivar Peoria. Given that thermal stress can stunt

growth in plants (Bharti and Khurana 1997, Grace et al. 1998),
and given that the lowest total biomass production occurred
in our tomato plants at 35 C this temperature may represent
heat stress.
The first step in iron metabolism is uptake, which requires
the reduction of Fe3 + to Fe2 + . The reduction capacity is
based on a reduction step at the cell surface and this is obligatory before chelate splitting and uptake of Fe2 + occurs
(Chaney et al. 1972). This ability to reduce extracellular Fe
compounds is due to an enzyme activity located on the plant
plasma membrane of the cell roots, called Fe (III)-chelate reductase (FeCH-R) (Bttger et al. 1991, Abada 1993). The uptake of Fe, and thus FeCH-R activity, is affected by a great
number of factors, such as pH and temperature (Hamlin et al.
1999). In our experiment with tomato roots, FeCH-R activity
differed significantly in response to the three temperatures
applied (Table 1). The highest FeCH-R value was recorded at
25 C, this temperature being optimal for the development of
tomato plants. However, at 35 C, FeCH-R activity was minimum, representing a reduction of 52 % with respect to 25 C
(Table 1). Our results agree with those of Hood and Mills
(1994), who observed that Fe uptake was lower at 36 C than
at 22 C. This indicates that high temperatures could be inhibiting FeCH-R activity.
The fact that the highest FeCH-R activity was recorded at
25 C would explain the highest concentrations, both in the
roots and the leaves, of total and free Fe at this temperature
(Table 1). On the contrary, 35 C resulted in lowest FeCH-R activities, as well as the lowest root and leaf concentration of total and free Fe (Table I).
In short, these results suggest that in tomato plants submitted to high temperature (35 C), FeCH-R activity sharply
declines because the incorporation of this micronutrient in the
plant diminished, lowering the total and free Fe concentrations in relation to values for tomato plants grown under optimal temperatures (25 C).

Table 1. Response of Fe metabolism and its bioindicators in tomato plants at three temperatures (10 C, 25 C and 35 C).
Organ

Temp

FeCH-R

Total Fe

Free Fe

Aco

GPX

CAT

FeSOD

Roots

10 C
25 C
35 C
Signif

2.4 0.31
4.4 0.22
2.1 0.24
**

3020 49
3787 46
2181 59
***

336 5
480 4
228 5
**

871 31
1351 30
451 29
***

37.5 0.5
51.6 0.9
18.7 1.1
***

22.9 0.5
30.6 0.3
10.7 0.4
***

Leaves

10 C
25 C
35 C
Signif

159 5
175 7
95.5 4
**

67.2 8
124.5 7
43.2 6
***

76.8 3.2
175 4.1
37.5 3.9
***

10.7 0.7
14.7 0.7
6.8 0.97
**

4.7 0.2
6.2 0.02
3.2 0.12
**

6.9 0.23
2.9 0.19
12 0.27
***

Data are means s. e. (n = 6). Levels of significance are represented by at * P < 0.05; ** at P < 0.01 and at *** P < 0.001 and ns: not significantly by
ANOVA at the 0.05 probability. FeCH-R = mol Fe reduced mg 1 protein min 1, Total and Free Fe = mol Fe g 1 DW; Aco = mol cis-aconitic acid
formed mg 1 protein min 1; GPX = mol guaiacol oxidized mg 1 protein min 1, CAT = mol H2O2 reduced mg 1 protein min 1; FeSOD = units FeSOD mg 1 protein min 1.

1068

Rosa M. Rivero et al.

Figure 1. Influence of temperature (10 C, 25 C, 35 C) on dry weight

of tomato and watermelon plants expressed as g d.w. per plant.

Figure 2. Influence of temperature (10 C, 25 C, 35 C) on H2O2 concentration in leaves of tomato and watermelon plants expressed as
mmol H2O2 (g f.w.) 1.

The activities of some metalloenzymes are used to diagnose Fe stress in plants, the most frequently used being GPX,
CAT and FeSOD (Iturbe-Ormaetxe et al. 1995, Lavon and
Goldschmidt 1999).
Another enzymatic activity also used to determine Fe
stress, although less frequently, is Aco. This enzyme, which
catalyses the dehydration of both isocitric and citric acids
from cis-aconitic acid, the reverse reaction, and the interconversion of citric and isocitric acids, contains iron-sulphur cluster and requires the addition of Fe for full activity (De Bellis et
al. 1993). In general, the relationship between the activity of
these bioindicators (GPX, CAT and Aco) and the Fe content
showed a positive and significant correlations in both leaves
and roots (LEAVES: GPX-total Fe, r = 0.921***; CAT-total Fe, r
= 0.823***; Aco-total Fe, r = 0.754**; ROOTS: GPX-total Fe, r =
0.901***; CAT-total Fe, r = 0.923***; Aco-total Fe, r = 0.798**),

since these enzymatic activities increase with the Fe content

(Table 1) (Iturbe-Ormaetxe et al. 1995, Lavon and Goldschmidt 1999).
In our experiments, Aco, GPX and CAT activities showed
the same trends under the three temperatures applied, reaching their highest activities at 25 C and lowest at 35 C. Aco
activity differed significantly, both in the leaves and roots, with
a reduction of 78 % at 35 C, with respect to the value at 25 C
(Table 1). The GPX and CAT activities showed the same behaviour, with a reduction of 54 % and 48 % in leaves, respectively, and of 65 % and 64 % in the roots, respectively, as temperature dropped from 35 C to 25 C (Table 1). The positive
correlation observed between Fe content and enzymatic activities indicates that these metalloenzymes are good bioindicators of the nutritional status of Fe.
Nevertheless, FeSOD activity followed a completely opposite trend from that of the other bioindicators described
above, with the highest activities appearing at 35 C for a
4-fold increase over values recorded at 25 C (Table 1). In
cold and heat environments the production of active oxygen
species (AOS), such as OH, O2 and H2O2 may be enhanced because the biosynthesis reactions are reduced and demand for ATP decreases (Scebba et al. 1998). In wheat, thermal stress has been demonstrated to prompt H2O2 over-production (Okuda et al. 1991). At 35 C, leaves of tomato plants
undergoing heat stress over-produce H2O2 (Fig. 2, P < 0.001),
which is synthesised principally by the FeSOD activity.
This could explain why this enzyme reaches its highest activity under thermal stress (35 C). This also explains why
FeSOD activity in tomato leaves is negatively related to the
total Fe concentration (r = 0.798**), despite the fact that
FeSOD activity depends on Fe levels. Finally, we observed
that the activities of SOD and CAT did not vary in the same
way. SOD catalyses the dismutation of O2 to H2O2 and O2
(McCord and Fridovitch 1969, Ushimaru et al. 2000). Increased SOD activity in spinach plants was found during
exposure to heat temperatures (Levine et al. 1994), and high
SOD activity has been associated with temperature stress in
plants where over-production of O2 is involved (Bowler et al.
1992, Rao et al. 1996). Therefore, SOD activity was induced to
a greater extent in the treatments in which the temperature
stress was strongest, as in 35 C for tomato plants and 10 C
for watermelon plants. The positive correlation found between
FeSOD activity and H2O2 concentration (FeSOD activityH2O2, r = 0.891***) could explain the high levels of H2O2 in
these plants. On the other hand, the high levels of H2O2 can
inhibit CAT activity (Willenkens et al. 1998), a situation that
would explain the inverse relationship between the enzymes
SOD and CAT.
Since H2O2 is a highly toxic compound, the first symptom
of H2O2 accumulation in plants is a reduction of the total biomass (Willenkens et al. 1997). Given that 35 C resulted in the
highest values for FeSOD activity (responsible for the H2O2
production) and the lowest for GPX and CAT activities (responsible for the elimination of H2O2), this temperature pro-

Effect of temperature on Fe metabolism

motes accumulation of this compound. This may be an explanation of the reduced total biomass in tomato plants at
35 C (Figs. 1 and 2).

Iron metabolism in watermelon plants

The watermelon requires more heat than does the tomato for
optimal development, 33 37 C (Veschambre and Zuang
1979, Maroto 1995). Therefore, under the three temperatures
applied (10 C, 25 C and 35 C), watermelon differed from the
tomato in its reaction, showing a cold stress at 10 C. The total
biomass was significantly lower (Fig. 1, P < 0.001), registering
a 58 % drop at to 35 C, the optimum growing temperature for
watermelon. Also, at 10 C the FeCH-R activity fell 38 % at
35 C (Table 2), showing a positive and significant correlation
with respect to total Fe (FeCH-R-Fe, r = 0.711**). The concentrations of total and free Fe also diminished at 10 C, by 43 %
and 65 % in leaves, respectively, and by 25 % and 53 % in
roots, respectively (Table 2).
The Aco, GPX and CAT activities followed a comparable
pattern (Table 2), with a reduction of 35 %, 50 % and 46 % in
leaves, respectively, and 43 %, 58 % and 37 % in roots, respectively, while showing a positive and significant relationship with the total Fe concentrations (LEAVES: GPX-total Fe, r
= 0.776**; CAT-total Fe, r = 0.821***; Aco-total Fe, r = 0.763**;
ROOTS: GPX-total Fe, r = 0.898***; CAT-total Fe, r = 0.791**;
Aco-total Fe, r = 0.865***).
The FeSOD activity, however, increased at 10 C by 61% at
35 C (Table 2). In addition, the relationship between FeSOD
activity and total Fe was negative and significant (r =
0.781**). Increased FeSOD activity in spinach plants was
found during exposure to high temperatures (Schner and
Krause 1990), and high FeSOD activity has been associated

1069

with heat in plants where over-production of O2 is involved

(Bowler et al. 1992, Rao et al. 1996).
Since FeSOD catalyses the dismutation of O2 to H2O2
and O2 (McCord and Fridovitch 1969, Ushimaru et al. 2000),
possibly in our experiment, at 10 C, H2O2 concentration rises
significantly (Fig. 2, P < 0.001) to almost 3.5-fold higher than
that found at 35 C. This appears to confirm that greater H2O2
accumulation may be due largely to the rise in FeSOD activity
at 10 C, given the positive relationship found between these
two parameters (SOD activity-H2O2, r = 0.912***). The above
results could explain the dry-weight reduction in watermelon
plants subjected to 10 C (Fig. 1), since one of the symptoms
of H2O2 accumulation is reduced foliar biomass (Fig. 2; Willenkens et al. 1997). The lower concentration of H2O2 resulted
at 35 C, coinciding with the highest foliar biomass (BiomassH2O2, r = 0.823***), results that appear to corroborate an
inverse relationship between the amount of biomass of a plant
and its foliar H2O2 concentration.
In short, the low temperature in the watermelon plants
caused the same effects as did high temperature in the
tomato plants, reducing: dry weigh per plant, FeCH-R activity,
total and free Fe concentrations, and Aco, GPX, and CAT
activities; while enhancing FeSOD activity and H2O2 concentrations provoked by enhanced FeSOD activity and reduced
GPX and CAT activities. The accumulation of H2O2 is concomitant with a reduction of biomass in watermelon plants.
With these results for the tomato and watermelon plants,
we conclude that, either excessive heat (tomato plants at
35 C) or excessive cold (watermelon plants at 10 C), with respect to the optimal growth temperature of the plant, reduces
uptake, distribution and accumulation of Fe while lowering the
activity of some related bioindicators (GPX, CAT and Aco). Finally, the high FeSOD activity in these plants could be explained by a defensive response to heat or cold stress.

Table 2. Response of Fe metabolism and its bioindicators in watermelon plants at three temperatures (10 C, 25 C and 35 C).
Organ

Temp

FeCH-R

Total Fe

Free Fe

Aco

GPX

CAT

FeSOD

Roots

10 C
25 C
35 C
Signif

3.3 0.26
4.1 0.21
5.2 0.29
**

2217 47
2401 54
2933 50
**

228 8
336 11
480 12
***

837 34
930 41
1464 46
***

25.1 0.6
30.3 0.9
58.7 1.2
**

17.2 0.7
21.4 0.4
27.4 0.5
**

Leaves

10 C
25 C
35 C
Signif

72 7.1
84 3.2
127 11
***

43 2.1
67 5.3
122 7
***

43.4 4.2
87.3 2.2
125 3.7
***

9.5 0.9
13.6 0.6
18.7 0.7
**

2.9 0.11
3.8 0.21
5.2 0.17
**

8.9 0.29
6.9 0.22
5.5 0.16
**

Data are means s. e. (n = 6). Levels of significance are represented by at * P < 0.05; ** at P < 0.01 and at *** P < 0.001 and ns: not significantly by
ANOVA at the 0.05 probability. FeCH-R = mol Fe reduced mg 1 protein min 1, Total and Free Fe = mol Fe g 1 DW; Aco = mol cis-aconitic acid
formed mg 1 protein min 1; GPX = mol guaiacol oxidized mg 1 protein min 1, CAT = mol H2O2 reduced mg 1 protein min 1; FeSOD = units FeSOD mg 1 protein min 1.

1070

Rosa M. Rivero et al.

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